Insulin-like growth factors (IGF's) have been isolated from various animal species, and are believed to be active growth promoting molecules that mediate the anabolic effects of such hormones as growth hormone and placental lactogen. As such, IGF's should be useful in the treatment and/or potentiation of various growth conditions and/or wound healing.
The designation "insulin-like growth factors" was chosen to express the insulin-like effects and insulin-like structure of these peptides. IGF's share nearly 50% amino acid homology with insulin and in three dimensional structure resemble proinsulin. Furthermore, by three dimensional modeling, the structures of IGF's are similar to proinsulin being a single chain peptide, cross-linked by three disulfide bridges and consisting of a B-chain-like aminoterminal part (B domain), a connecting peptide (C domain), and an A-chain-like part (A domain). In addition, a carboxyl-terminal extension not found in proinsulin is present (D domain). Recent studies also report the presence of yet another carboxyl-terminal extension not found in proinsulin which has been given an E domain designation. The E domain peptide has thus far been identified in association with rat and human IGF-II. Hylka et al. (1985) and Zumstein et al. (1985). To date, several classes of IGF's have been identified. These include IGF-I (Somatomedin C), IGF-II, Somatomedin A, and a mixture of peptides called multiplication-stimulating activity (MSA). This heterologous group of peptides exhibit important growth-promoting effects in vitro, Daughaday (1977); Clemmons and Van Wyk (1981a), and in vivo, van BuulOffers and Van den Brande (1980), Schoenle et al. (1982).
Two human IGF's have been characterized. These are IGF-I, comprising a 70 amino acid basic protein, Rinderknecht and Humbel (1978a), Rubin et al. (1982), and IGF-II, comprising a 67 amino acid neutralpeptide, Rinderknecht and Humbel (1978b); Marquardt and Todaro (1981). Whereas the complete amino acid sequences have only been determined for rat and human IGF-I and IGF-II, Humbel, R. E. (1984), a high degree of homology and/or cross-reactivity has been shown by radioimmunoassay and/or radioreceptor assay to exist among IGF-I's and among IGF-II's from different species. Wilson and Hintz (1982).
Circulating levels of these peptides appear to be under the control of growth hormone to a greater or lesser extent with IGF-I being controlled to a greater extent than IGF-II. IGF-I, for example, plays a fundamental role in postnatal mammalian growth as a major mediator of growth hormone action. See Copeland et al. (1980) and Schoenle et al. (1982).
Vassilopoulou-Sellin and Phillips (1982) have estimated, using molecular sieve chromatography, that IGF-I activity, assayed both in vitro and in vivo, extracted from rat liver has a higher molecular weight (approximately 30 kilodaltons) than activity extracted from plasma (approximately 8 kilodaltons). The authors suggested that the higher molecular weight material may represent an IGF-I precursor. The authors also demonstrated that metabolic regulation of the higher molecular weight rat liver IGF-I was similar to rat serum-derived IGF-I. Recently, Zumstein et al. (1985) isolated a variant pro-form of IGF-II from human serum which they demonstrated to contain IGF.about.like activity in vitro.
Because of the potential bioactivity and utility of high molecular weight, precursor IGF-I proteins in the treatment and/or potentiation of various growth conditions, the amino acid sequence and DNA coding sequence of such a precursor protein has long been sought.
Jansen et al. (1983), provided an amino acid sequence derived from a human IGF-I cDNA clone which supports the suggestion of a larger IGF-I precursor. See also Netherlands Patent Application No. 8302324, published Jan. 16, 1985. The cDNA disclosed by Jansen, however, did not provide sufficient DNA sequence information to teach the precise translational start of the suggested precursor protein. Additionally, Jansen et al. (1983) and Netherlands Patent Application No. 8302324 provide no evidence or teaching that the cDNA sequence is or can be expressed (e.g., that the suggested precursor protein is produced).
Indeed, very little is known about IGF-I biosynthesis. Preliminary studies suggest that only one human IGF-I gene exits per haploid genome. Ullrich et al. (1984); Brissenden et al. (1984); and Tricoli et al. (1984). Studies of IGF-I biosynthesis have been hampered by a very low IGF-I content in tissue, Vasilopoulou-Sellin and Phillips (1982) and because, in contrast to IGF-II, no cultured cell lines have been identified which elaborate significant quantities of this peptide. Clemmons and Van Wyk (1981b); Clemmons and Shaw (1983). Additionally, neither the complete human IGF-I gene has been isolated nor the complete DNA sequence determined. Preliminary studies by Bell et al. (1985) suggest that the human IGF-I gene is at least 35 kilobases (kb) in length, of which only 210 base pairs (bp) encodes mature human IGF-I. The studies by Bell et al. (1985) also suggest that the human IGF-I gene contains only four exons, which together encode a single precursor IGF-I protein. The large size (e.g. greater than 35 kb) and complexity of this gene relative to the mature IGF-I coding sequences has made both isolation and identification of the complete genomic DNA sequences extremely difficult. Isolation of a genomic IGF-I clone (e.g. IGF-I gene) would greatly facilitate studies of IGF-I biosynthesis and provide a means for identifying and producing precursor, mature and/or intermediate IGF-I species and allelic variants thereof. A genomic clone thus facilitates determining which proteins are believed to be active growth promoting peptides.
Accordingly, it is an object of this invention to provide a highly purified gene and/or synthetic DNA sequences encoding an IGF-I precursor protein (preproinsulin-like growth factor-I), and/or peptide fragments thereof and useful in making such a protein and/or fragments.
It is another object of this invention to provide processes utilizing such DNA in the production of such proteins and peptides.
Another object of this invention is to provide the amino acid sequence of a novel preproinsulin-like growth factor-I and methods using such a protein or fragments thereof to promote desirable growth or functionality of cells in animals.